CN106497863B - A kind of separation, purifying and the cultural method of cornea of rats endothelial cell - Google Patents
A kind of separation, purifying and the cultural method of cornea of rats endothelial cell Download PDFInfo
- Publication number
- CN106497863B CN106497863B CN201510560497.7A CN201510560497A CN106497863B CN 106497863 B CN106497863 B CN 106497863B CN 201510560497 A CN201510560497 A CN 201510560497A CN 106497863 B CN106497863 B CN 106497863B
- Authority
- CN
- China
- Prior art keywords
- cornea
- cell
- endothelial cell
- rats
- dmem
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of method of separation, purifying and the culture of cornea of rats endothelial cell, belongs to cell biology；It obtains individual cells the following steps are included: the separation of (a) cornea tissue sample；(b) immunomagnetic beads for preparing specific adsorption endothelial cell, purify endothelial cell from isolated individual cells；(c) cell culture fluid optimized cultivates cornea of rats endothelial cell.The separation method of cornea of rats endothelial cell provided by the invention, easy to operate, cell yield and high survival rate；Cornea of rats endothelial cell purification process provided by the invention, easy to operate, yield and purifying rate are very high；Cornea of rats Endothelial cell culture method provided by the invention, cell Proliferation block, period be short and high financial profit.
Description
Technical field
The present invention relates to the methods of a kind of separation of the endothelial cell of rat, purifying and culture, belong to cell biological
Field.
Background technique
Endothelial cell is morphologic complete and functionally sound is the index for clinically evaluating cornea function,
It is the standard of preservation of cornea performance rating.And various disease of cornea, surgery damage and drug toxicity etc. can all cause in cornea
Chrotoplast is impaired, when damage is more than its physiological compensation, keratopathy will occur, will lead to corneal blindness when serious.For this purpose, seeing
It examines drug and the active influence of some other factor corneal endothelium all be unable to do without the experiment in vitro of endothelial cell,
And good external endothelial cell separation and cultural method are then the first steps of experiment in vitro, for the pharmacology of cornea
The research of, pathology and corneal transplantation has great importance.
Cornea tissue from people is few, and the division growth ability of its endothelial cell is low, only in embryo
Phase has certain proliferative capacity.Zoopery proves that monkey and rabbit corneal endothelium only have limited division growth in vitro
Ability, and the endothelial cell of mouse all has good proliferative capacity under conditions of wound stimulation or in vitro culture.For this purpose,
The eyeball of rat is the vital tissue source for being used in vitro culture endothelial cell.
Currently, the primary culture in vitro of endothelial cell includes: tissue block adherent method, enzyme digestion (pancreatin, clostridiopetidase A
With dispase enzyme), microdissection etc., but microdissection is complicated for operation；When enzyme digestion needs strict control enzymic digestion
Between and enzyme concentration, to prevent damage of the enzyme to cell, and it is slower with growth rate to organize adherent rule cell to move out.And it organizes
During Dissociated cell culture, it often will appear the problem of stroma cell pollutes.For this purpose, how to pass through relatively simple method
The endothelial cell that quantity is more, activity is good and with high purity is obtained, is always that progress basic research and clinical research are urgently to be resolved
Major issue.
Summary of the invention
More, activity that the purpose of the present invention is to provide a kind of acquisition quantity is well and the cornea of rats endothelial cell of purity is high divides
From, purifying and cultural method.
The separation of cornea of rats endothelial cell, purifying and cultural method provided by the invention, it includes the following steps:
A, the separation of cornea tissue sample: under rat anesthesia disinfection, rat eye is first taken out, removes cornea, then use
Disinfection blade shreds cornea, finally with digestion enzymatic treatment cornea tissue, obtains individual cells.
Wherein, the rat eye comes from the male SD rat of 4 week old, and weight quality is 150 ± 25g；
The removing cornea is the annular clip Full-thickness corneal along 1mm outside corneal limbus；
The digestion enzymatic treatment cornea tissue, method particularly includes: the dispase of 0.25% (m/v) preheated with 37 DEG C
The clostridiopetidase A I of enzymic digestion 30-40min, pancreatin and 0.1% (m/v) that 0.05-0.1% (m/v) is then added digest 5-10min,
Add complete medium to terminate digestion, blow and beat digestive juice repeatedly, filtrate is collected in the filtering of 100 mesh holes.Filtrate by 1500rpm from
Heart 5min, discards supernatant, and DMEM/F12 complete medium is then added, and suspension cell precipitates again, single after collection angle membrane digestion
The cell liquid of a cell.
B, the purifying of endothelial cell: the immunomagnetic beads of preparation specific adsorption endothelial cell, specific immunity
Magnetic bead purifies the cell liquid of individual cells in step a.
Wherein, the preparation specific immunity magnetic bead, method particularly includes: by the immunomagnetic beads of goat antirabbit in DMEM/
Clean 3 times in F12 basal medium, then in monoclonal PECAM-1 antibody 4 DEG C be incubated overnight, it is then complete in DMEM/F12
It cleans 3 times, is finally resuspended in DMEM/F12 complete medium in full culture medium；
The endothelial cell purifying, method particularly includes: it is individually thin that the immunomagnetic beads prepared are added to step a
In the cell liquid of born of the same parents, 4 DEG C of rotation jogs are incubated for 30-60min, then with cleaning immunomagnetic beads 2- in DMEM/F12 complete medium
4 times, the purity of cell is detected by low cytometric analysis, is finally added into DMEM/F12 complete medium and is cultivated increasing
It grows.
C, optimizing culture-medium: preparing cornea of rats endothelial lysis liquid, addition basic fibroblast growth because
Son, the complete medium of the cornea of rats endothelial cell of configuration.
Wherein, the preparation of the cornea of rats endothelial lysis liquid, method particularly includes: by step b after purification
Cornea of rats endothelial cell is cultivated in DMEM/F12 complete medium, and is passed on, when cell quantity reaches culture bottle area
When 80%, cell pancreatin is digested, and cell pyrolysis liquid is obtained by multigelation method, is obtained finally by BCA method
The protein concentration of lysate.
Wherein, contain following ingredient: 10-100mg cell cracking for every liter of complete medium of the cornea of rats endothelial cell
Liquid, 1-7.5mg basic fibroblast growth factor, 100mg fetal calf serum, remaining be DMEM/F12 basal medium.
Further, contain following ingredient for every liter of complete medium of the cornea of rats endothelial cell: 80mg cell is split
Solve liquid, 5.5mg basic fibroblast growth factor, 100mg fetal calf serum, remaining be DMEM/F12 basal medium.
Bright spot of the invention and the utility model has the advantages that
1. the present invention by simply shredding cornea tissue, then obtains endothelial cell, the party by enzymic digestion
Method is easy to operate, and can obtain endothelial cell a large amount of and with excellent activity.
2. specific immunity magnetic bead prepared by the present invention specific adsorption endothelial cell, purifying rate can reach well
To 90% or more.
3. can promote a large amount of proliferation of cell the present invention provides a kind of endothelial cell culture medium, greatly shorten
The incubation time of cell.
Detailed description of the invention
Fig. 1 has the fluidic cell of no added streaming antibody to compare for the cornea of rats endothelial cell obtained after magnetic bead sorting
Figure；Wherein A is the fluidic cell figure for not adding streaming antibody；B is the cell that obtains after magnetic bead sorting, the stream after streaming antibody is added
Formula cytological map；
Fig. 2 be cornea of rats endothelial cell in heterogeneity cell culture medium for 24 hours after cell growthform；Wherein A
For the cytological map (× 100) after DMEM/F12 complete medium culture for 24 hours；B is that the DMEM/F12 of the addition bFGF factor is trained completely
Cytological map (× 100) after supporting base culture for 24 hours；C be add cell pyrolysis liquid DMEM/F12 complete medium culture for 24 hours after
Cytological map (× 100)；D is the cell after the DMEM/F12 complete medium culture for 24 hours of the addition bFGF factor and cell pyrolysis liquid
Scheme (× 100)；
Fig. 3 is that cornea of rats endothelial cell is trained in the DMEM/F12 complete medium of the addition bFGF factor and cell pyrolysis liquid
Cytological map (× 400) after supporting 4d.
Specific embodiment
The contents of the present invention are further specifically illustrated combined with specific embodiments below and are illustrated, but following embodiment is only
Illustrative, it is not used in and limits the scope of the invention.
The present invention is object in 150 ± 25g with 4 weeks or so SD rats, male, weight quality, and it is thin to obtain corneal endothelium
Born of the same parents' is tissue-derived.Concrete operations are as follows:
Step 1: removing cornea of rats tissue, and obtain individual cells
Give rats by intraperitoneal injection yellow Jackets anesthesia, with the ethanol disinfection of 75% (v/v), immediately aseptically with
Curved tweezers take out the eyeball of rat, in penicillin (100U/ml)-streptomysin (100 μ g/ml) of the working concentration containing cell culture
Excess tissue is removed in sterile saline, and is cleaned for several times, is subsequently placed in clean work station, eyeball is moved into sterilized petri dishes
In, in the annular clip Full-thickness corneal at 1mm outside corneal limbus, in the phosphate buffer (PBS, pH 7.4,10mM) that 37 DEG C incubate
Middle rinsing 3 times.
Cornea is shredded as far as possible with disinfection blade, is sufficiently mixed uniformly, is digested with digestive ferment, specific method
Are as follows: then the dispase enzymic digestion 30-40min of 0.25% (m/v) preheated with 37 DEG C is added and contains 0.05-0.1% (m/v) pancreas
The mixture slaking liquid of enzyme and 0.1% (m/v) clostridiopetidase A I digest 5-10min, and complete medium is added to terminate digestion, and piping and druming disappears repeatedly
Change liquid, filtrate is collected in the filtering of 100 mesh holes.The filtrate that above-mentioned three kinds of methods obtain can be centrifuged 5min by 1500rpm, abandon
Supernatant is removed, DMEM/F12 complete medium (the DMEM/F12 basal medium containing 10% fetal calf serum) is then added and hangs again
Floating cell precipitation.
Step 2: specific immunity magnetic bead purifies endothelial cell from isolated individual cells
The preparation of specific immunity magnetic bead: the immunomagnetic beads of the goat antirabbit of purchase are clear in the DMEM/F12 of serum-free
Wash 3 times, then in monoclonal PECAM-1 antibody 4 DEG C be incubated overnight, then cleaned 3 times in DMEM/F12 complete medium,
It is finally resuspended in DMEM/F12 complete medium.
Endothelial cell purifying: the immunomagnetic beads prepared are added in step (a) after passing through digestion enzymic digestion
In DMEM/12 complete medium in the cell liquid of resuspension, 4 DEG C of rotation jogs are incubated for 30-60min, and then DMEM/F12 is complete
Culture medium cleans immunomagnetic beads 2-4 times, and the purity of cell is detected by low cytometric analysis, is finally added into complete culture
In base, it is placed in the CO of 37 DEG C, 5% (v/v)2Proliferation is cultivated in the incubator of saturated humidity.
The purity of the endothelial cell obtained after flow cytomery digestion enzymic digestion: (1) cell is collected into
In the centrifuge tube of 15ml, 1500rpm is centrifuged 5min, abandons suspension；(2) cell is washed with PBS, 1500rpm is centrifuged 5min；(3) add
Enter the methanol of -20 DEG C of pre-coolings, piping and druming is uniform；(4) cell is washed with PBS, 1500rpm is centrifuged 5min；(5) it is diluted with 200 μ l single
It clones PECAM-1 antibody and cell is resuspended, 37 DEG C are protected from light incubation 1h；(6) it is washed cell 2 times with PBS, 1500rpm is centrifuged 5min；
(7) cell, upper machine testing is resuspended with 400 μ l PBS.
Reach 90% or more by magnetic bead sorting endothelial cell purifying rate as shown in Figure 1:.
Step 3: the cell culture fluid of optimization, cultivates cornea of rats endothelial cell
The preparation of cornea of rats endothelial lysis liquid: 2ng/mL is being contained by cornea of rats endothelial cell after purification
Basic fibroblast growth factor (bFGF) DMEM/F12 complete medium in cultivate, reach 80% or more to cell
It when degrees of fusion, is passed on 1: 2 amount, after passing number generation, when cell quantity reaches the 80% of culture bottle area, by cell pancreas
Enzymic digestion is got off, and the fetal calf serum culture medium being added containing 10% terminates digestion, and then 1500rpm is centrifuged 5min and removes supernatant, adds
Enter DMEM/F12 basal medium and gently blow and beat cell precipitation, cell is made to suspend, then 1500rpm centrifugation 5min removes supernatant, it is above
It states similarly suspension, centrifugal method to be repeated 3 times to remove fetal calf serum ingredient therein, is eventually adding enough basal mediums
Endothelial cell is resuspended in DMEM/F12, places it in and freezes 10-20min in -80 DEG C, then places 37 DEG C of environment rapidly
Middle defrosting is so repeated 3 times so that cornea of rats endothelial cell sufficiently cracks, in 0.22 μm of filter filtering removal lysate
Cell fragment, this is cornea of rats endothelial lysis liquid, leaves and takes 0.5mL sample BCA kit detection protein content.
The optimization of cornea of rats Endothelial cell culture base: will grow to the 3rd generation cornea of rats endothelial cell of 80% degrees of fusion,
It is digested with pancreatin, DMEM/F12 complete medium is added and terminates digestion, 1000r/min is centrifuged 5min, is made 2 × 104cells/
The cell suspension of mL, by 500 holes μ L/ be inoculated with 24 orifice plates in, culture discard culture solution afterwards for 24 hours, with PBS rinse for several times, respectively plus
Enter in following 4 kinds of different culture mediums:
(1) in the DMEM/F12 complete medium for not adding any additive；
(2) in the DMEM/F12 complete medium containing 2-15ng/ml bFGF；
(3) in the DMEM/F12 complete medium containing the white 10-100 μ g/ml of cornea of rats endothelial lysis liquid eggs；
(4) contain -100 μ g/ml of cell pyrolysis liquid protein 10 and basic fibroblast growth factor (bFGF) 1-
In the DMEM/F12 complete medium of 7.5ng/ml.
Every kind of culture solution sets 6 holes, and the cell liquid of every empty 500 μ L observes cell life after continuing culture for 24 hours under the microscope
Long form.Continue to cultivate cell, and observes cell growth condition.
It is as shown in Figure 2: in the DMEM/F12 containing -100 μ g/ml and bFGF 1-7.5ng/ml of cell pyrolysis liquid protein 10
Cell Proliferation is apparently higher than other group of cell in complete medium, and the form of cell is all very good.
As shown in Figure 3: endothelial cell is containing -100 μ g/ml of cell pyrolysis liquid protein 10 and bFGF 1-7.5ng/
After cultivating 4d in the DMEM/F12 complete medium of ml, the basic confluent cultures ware of cell.
Claims (3)
1. a kind of method of purifying and the culture of cornea of rats endothelial cell, which is characterized in that it the following steps are included:
A, the preparation of cornea individual cells: shredding cornea with disinfection blade, finally with digestion enzymatic treatment cornea tissue, obtains single
Cell；
The digestion enzymatic treatment cornea tissue, method particularly includes: the dispase enzyme of 0.25% (m/v) preheated with 37 DEG C disappears
Change 30-40min, the clostridiopetidase A I of pancreatin and 0.1% (m/v) that 0.05-0.1% (m/v) is then added digests 5-10min, adds
Full culture medium terminates digestion, blows and beats digestive juice repeatedly, and filtrate is collected in the filtering of 100 mesh holes；Filtrate is centrifuged by 1500rpm
5min is discarded supernatant, and DMEM/F12 complete medium is then added, and suspension cell precipitates again, single after collection angle membrane digestion
The cell liquid of cell；
B, the purifying of endothelial cell: the immunomagnetic beads of preparation specific adsorption endothelial cell, specific immunity magnetic bead
The cell liquid of individual cells in step a is purified；
C, optimizing culture-medium: preparing cornea of rats endothelial lysis liquid, adds basic fibroblast growth factor, matches
The complete medium of cornea of rats endothelial cell processed；
Every liter of complete medium of the cornea of rats endothelial cell contains following ingredient: 10-100mg cell pyrolysis liquid, 1-7.5mg
Basic fibroblast growth factor, 100mg fetal calf serum, remaining be DMEM/F12 basal medium.
2. the method for purifying and the culture of cornea of rats endothelial cell according to claim 1, which is characterized in that the step
The purification process of b are as follows: clean the immunomagnetic beads of goat antirabbit 3 times in DMEM/F12 basal medium, then in monoclonal
It is incubated overnight for 4 DEG C, is then cleaned 3 times in DMEM/F12 complete medium, the filtrate collected after digestion in PECAM-1 antibody
In, 4 DEG C of rotation jogs are incubated for 30-60min, then clean magnetic bead 2-4 times with DMEM/F12 complete medium, are finally added into
In DMEM/F12 complete medium, it is placed in 37 DEG C, cultivates proliferation in the incubator of the CO2 saturated humidity of 5% (v/v).
3. the method for purifying and the culture of cornea of rats endothelial cell according to claim 1, which is characterized in that the rat
Every liter of endothelial cell complete medium contains following ingredient: 80mg cell pyrolysis liquid, 5.5mg basic fibroblast growth
The factor, 100mg fetal calf serum, remaining be DMEM/F12 basal medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510560497.7A CN106497863B (en) | 2015-09-07 | 2015-09-07 | A kind of separation, purifying and the cultural method of cornea of rats endothelial cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510560497.7A CN106497863B (en) | 2015-09-07 | 2015-09-07 | A kind of separation, purifying and the cultural method of cornea of rats endothelial cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106497863A CN106497863A (en) | 2017-03-15 |
| CN106497863B true CN106497863B (en) | 2019-10-11 |
Family
ID=58287931
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510560497.7A Active CN106497863B (en) | 2015-09-07 | 2015-09-07 | A kind of separation, purifying and the cultural method of cornea of rats endothelial cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106497863B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108728405A (en) * | 2017-04-21 | 2018-11-02 | 江苏齐氏生物科技有限公司 | A kind of Isolation Methods of Adult Rat Ventricular Cardiomyocytes |
| CN112522179A (en) * | 2020-12-23 | 2021-03-19 | 江苏艾尔康生物医药科技有限公司 | Separation culture method of corneal endothelial cells |
| CN115197913A (en) * | 2021-04-13 | 2022-10-18 | 江苏齐氏生物科技有限公司 | Primary corneal endothelial cell culture solution and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001063281A1 (en) * | 2000-02-23 | 2001-08-30 | Musc Foundation For Research Development | Methods of screening for compounds that modulate blood vessel formation |
| CN1898377A (en) * | 2003-10-10 | 2007-01-17 | 细胞生物工程公司 | Human corneal endothelial cells and methods of obtaining and culturing cells for corneal cell transplantation |
| CN101597592A (en) * | 2009-06-11 | 2009-12-09 | 山东省眼科研究所 | Human corneal endothelial cell culture solution and its production and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877955A (en) * | 2015-06-12 | 2015-09-02 | 北京农学院 | Method for separating and purifying rat dermal microvascular endothelium cells |
-
2015
- 2015-09-07 CN CN201510560497.7A patent/CN106497863B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001063281A1 (en) * | 2000-02-23 | 2001-08-30 | Musc Foundation For Research Development | Methods of screening for compounds that modulate blood vessel formation |
| CN1898377A (en) * | 2003-10-10 | 2007-01-17 | 细胞生物工程公司 | Human corneal endothelial cells and methods of obtaining and culturing cells for corneal cell transplantation |
| CN101597592A (en) * | 2009-06-11 | 2009-12-09 | 山东省眼科研究所 | Human corneal endothelial cell culture solution and its production and application |
Non-Patent Citations (1)
| Title |
|---|
| Involvement of Endothelial PECAM-1/CD31 in Angiogenesis;Horace M. DeLisser等;《American Journal ofPathology,》;19970930;第151卷(第3期);671-676 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106497863A (en) | 2017-03-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102127522B (en) | Human umbilical mesenchymal stem cell and preparation method thereof | |
| CN104164403B (en) | Method for extracting and culturing adipose-derived stem cells | |
| CN104726406B (en) | It is a kind of to induce the method that dental pulp Derived from Mesenchymal Stem Cells is nerve cell | |
| CN106801032B (en) | Construction method of human amniotic epithelial stem cell bank | |
| WO2016049986A1 (en) | Method for separating umbilical cord mesenchymal stem cells | |
| CN102344906B (en) | Hair follicle stem cell separation culture method | |
| CN104450611A (en) | Primary separation and culture method of human amniotic mesenchymal stem cells | |
| CN105950550A (en) | Mesenchymal stem cell serum-free medium and cell isolation and cultivation methods | |
| CN106834224A (en) | It is a kind of to set up the method that human pluripotent stem cells are induced to differentiate into mature blood cell | |
| CN106497863B (en) | A kind of separation, purifying and the cultural method of cornea of rats endothelial cell | |
| CN107385517A (en) | The construction method of mesenchyma stem cell | |
| CN105543164A (en) | Primary isolated culture method for dairy cow mammary epithelial cells | |
| CN104762257A (en) | Method for preparing mesenchymal stem cell from umbilical cord | |
| JP2018526025A (en) | Method for preparing olfactory nerve sheath cells | |
| CN106244533A (en) | The primary separation method of gingiva mescenchymal stem cell | |
| CN103396985A (en) | Method for inducing differentiation of human umbilical cord mesenchymal stem cells into hepatocytes and applications | |
| CN101831403A (en) | Method for amplifying mesenchymal stem cells of human umbilical cord and placenta in vitro | |
| CN104031881B (en) | A kind of isolated culture method of mankind's olfactory mucosa mescenchymal stem cell | |
| CN104403988B (en) | A kind of inducing mouse embryonic stem cell breaks up the method for inner ear hair cells | |
| CN101597592A (en) | Human corneal endothelial cell culture solution and its production and application | |
| CN106834217A (en) | A kind of method for promoting human amnion membrane amplification in vitro and application | |
| CN113186155B (en) | High-efficiency culture method of primary cells of sheep embryonic skeletal muscle | |
| CN102796701A (en) | Method for amplifying human umbilical cord mesenchymal stem cells through in-vitro 3D stent-free suspension cultivation | |
| CN110387351A (en) | A kind of isolation and culture method of human retina Muller cell | |
| CN109439628A (en) | Limbal stem cell primary culture method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |